This invention relates to new assay methods for monitoring the concentration in bodily fluids of immunosuppressants affecting gene expression, e.g., cyclosporins, such as cyclosporin A or cyclosporin G; ascomycins, such as FK-506; and rapamycins, e.g., rapamycin; using a reporter gene assay, e.g., an IL-2 reporter gene assay for immunosuppressive cyclosporins and ascomycins, or a c-jun reporter gene assay for immunosuppressive rapamycins.
Cyclosporins comprise a class of structurally distinct, cyclic, poly-N-methylated undecapeptides, generally possessing immunosuppressive, anti-inflammatory, anti-viral and/or anti-parasitic activity, each to a greater or lesser degree. The first of the cyclosporins to be identified was the fungal metabolite Cyclosporin A, or Ciclosporin, and its structure is given in The Merck Index, 11 th Edition; Merck & Co., Inc.; Rahway, N.J., USA (1989) under listing 2759. Later cyclosporins to be identified are cyclosporins B, C, D and G which are also listed in the Merck Index under listing 2759. A large number of synthetic analogues are also known and representative examples are disclosed in EP 296 123, EP 484 281, and GB 2222770. Cyclosporin A and its structurally similar analogues and derivatives are generally referred to as "cyclosporins" for the purposes of this specification.
Rapamycin is a macrolide immunosuppressant that is produced by Streptomyces hygroscopicus and which has been found to be pharmaceutically useful in a variety of applications, particularly as an immunosuppressant for use in the treatment and prevention of organ transplant rejection and autoimmune diseases. The structure of rapamycin is given in Kesseler, H., et al.; 1993; Helv. Chim. Acta; 76: 117. Large numbers of derivatives of rapamycin have been synthesized, including for example 40-O-alkylated derivatives such as 40-O-(2-hydroxy)ethyl-rapamycin (WO 94/09010), certain acyl and aminoacyl-rapamycins (e.g., U.S. Pat. No. 4,316,885, U.S. Pat. No. 4,650,803, and U.S. Pat. No. 5,151,413), 27-desmethyl-rapamycin (WO 92/14737), 26-dihydro-rapamycin (U.S. Pat. No. 5,138,051), certain pyrazole derivatives (U.S. Pat. No. 5,164,399), certain alkoxyester derivatives (U.S. Pat. No. 5,233,036), and numerous others. Rapamycin and its structurally similar analogues and derivatives are termed collectively as "rapamycins" in this specification.
Ascomycins, of which FK-506 is the best known, are another class of generally immunosuppressive macrolides. FK506 is a macrolide immunosuppressant that is produced by Streptomyces tsukubaensis No 9993. The structure of FK506 is given in the appendix to the Merck Index, as item A5. A large number of related compounds which retain the basic structure and immunological properties of FK506 are also known. These compounds are described in various publications, for example EP 184162, EP 315973, EP 323042, EP 423714, EP 427680, EP 465426, EP 474126, WO 91/13889, WO 91/19495, EP 484936, EP 532088, EP 532089, WO 93/5059 and the like. Ascomycin, FK-506 and their structurally similar analogues and derivatives are termed collectively "ascomycins" in this specification.
Due to their extremely useful pharmaceutical properties, cyclosporins (Cyclosporins A and G in particular), rapamycins (e.g., rapamycin and 40-O-(2-hydroxy)ethyl-rapamycin) and ascomycins (e.g., FK-506) have wide application in, for example the prevention of transplant rejection and in the treatment of auto-immune diseases. However these compounds have side effects at higher doses and therefore their concentration in the blood must be kept within certain therapeutic ranges. Bioavailabilities and metabolic conversion rates tend to be patient specific and hence dosaging is patient specific. It is thus desirable to monitor the blood levels of such drugs and to adjust the dosage to obtain optimum blood levels of the drug thereby maximizing immunosuppression and minimizing adverse side effects.
Up until now, the method of choice for measuring blood levels of immunosuppressants has been using immunoassays with monoclonal antibodies (MAbs) specific for the immunosuppressants to be tested, or when mAb assays have not been available or practical, using HPLC. The immunoassay method has several very important drawbacks. First, different MAbs have different levels of affinity to the immunosuppressants and to their metabolites. Thus, it is difficult to make meaningful comparisons of data obtained by clinicians using different MAbs. Second, MAbs cannot distinguish between pharmacologically active and pharmacologically inactive metabolites. For example, a MAb that is very specific for the parent drug may not bind to a pharmacologically active metabolite and would thus understate the blood levels of immunosuppressant and lead to a serious risk of overdosage. On the other hand, a less specific MAb binds pharmacologically inactive metabolites as well as active metabolites, thus overstating the blood levels of immunosuppressant. HPLC likewise provides no information as to the immunosuppressive versus nonimmunosuppressive metabolite levels, and it is furthermore a relatively time-consuming and difficult method.
The problems with immunoassay methods have been particularly acute in the case of FK-506. A large contribution of the immunosuppressive activity of FK-506 derives not from FK-506 Per se, but from various unidentified immunosuppressive metabolites of FK-506. For example, it was recently reported in a study of thirteen transplant patients, that the ratio between FK-506 and one of its unidentified immunosuppressive metabolites detectable by HPLC was 1:10 in blood serum. Moreover, the metabolism of FK-506 is very erratic and highly variable from patient to patient. Finally, because of the very high potency of FK-506, it is necessary to achieve accurate measurements of the drug at extremely low concentrations, e.g., low nanomolar and sub-nanomolar concentrations. Commentators have pointed out the wide variability between observed levels of FK-506 using various immunoassays and the need to develop more reliable and consistent assays.
There is thus a clear and unfulfilled need to provide a monitoring system capable of accurately detecting blood levels of immunosuppressant drugs and their pharmacologically active metabolites. The present invention meets that need by providing a simple and sensitive method of monitoring blood levels of immunosuppressant using a reporter gene assay. The assay system can be applied to measure concentrations of any drug that influences expression of a gene in a dose dependant manner, but has been found to be particularly sensitive to and suitable for measuring concentrations of ascomycins, especially FK-506.
Although some reporter gene assays have been previously described in the literature, the use of such assays for diagnostic assays is new and inventive, particularly as it was not previously shown that the degree of inhibition of the assay could be correlated with the concentration of immunosuppressant sufficiently closely to permit highly accurate measurements of subnanomolar concentrations of immunosuppressants, thereby filling the long felt need for accurate measurement of very small concentrations of immunosuppressive substances in bodily fluids.